Discovery of the genetic basis of inherited arrhythmia syndromes (IAS) has had significant impact on the diagnosis and clinical treatment of patients. As experiments of nature it has also contributed basic insights into the normal and abnormal function of ion channels, signaling pathways and mechanisms of arrhythmia. The cardiac Na current INa underlies excitability in heart; it is carried by the cardiac sodium channel NaV1.5, which is part of a sodium channel complex (SCC) composed of >29 Na channel interacting proteins (SCIPs). Of the established IAS genes 40% are in SCIPs that affect INa; much discovery remains to further our understanding of such IAS as long QT syndrome (LQTS), Brugada Syndrome (BrS), and Sudden Infant Death Syndrome (SIDS). This new application requests funds in part for a gene discovery project that in the past has contributed to establishing the genetic basis of ten arrhythmia syndromes. Mutations in two more SCIPs (SAP97 and Nedd4.2L), not previously known to be arrhythmia genes, will be investigated for plausible pathogenicity of BrS through INa dysfunction. Additional screening of inherited arrhythmia cohorts will continue and new discoveries investigated. Beyond discovery, this project addresses mechanisms by which mutations in SCIPs cause dysfunction of INa. This application builds on previously published work showing that enhanced direct S-Nitrosylation (SNO) of NaV1.5 underlies increased late INa for both LQT9 and LQT12 through loss of function of the SNO inhibitory actions of caveolin-3 and the alpha1-syntrophin/plasma-membrane Ca2+ pump complexes, respectively. Aim 1 will address important questions about causes of increased late INa by direct SNO of NaV1.5 cysteine residues and will include novel hypotheses about specific SNO sites on NaV1.5, roles for denitrosylation regulation of INa, and if SNO mechanisms cause late INa in eight representatives inherited and acquired disorders. Aim 2 will investigate mechanisms of SNO of the E3 ubiquitin Ligase Nedd4.2L in regulating INa density. Some LQT9 and LQT12 mutations also affected peak INa, this aim will investigate the hypothesis that Nedd4.2L is inactivated by SNO at a specific cysteine site decreasing ubiquitination of NaV1.5 and thereby increasing INa. Nedd4.2L exists in both short and long splice variants with the long variant regulated by Ca2+. Our preliminary data show both variants are present in human heart with the long form dominant. After determining the composition of Nedd4.2L variants in the SCC we will determine Ca2+ and SNO regulation of INa density. The appreciation of the role of SNO of the SCC in regulating excitability is just beginning. SNO is a general cell phenomenon and unraveling the mechanisms by which SNO of NaV1.5 and SCIPs regulate INa has particular significance for understanding basic mechanisms of specificity of cell signaling by establishing the principle of local control by co-localization of key membrane proteins and substrates. SNO regulation of INa will likely have applicability to arrhythmogenesis in acquired heart disease, such as heart failure and ischemia, where SNO signaling is known to be affected.